Immunofluorescence / Confocal Microscopy Protocol
实验概要
Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. Immunofluorescence is a widely used example of immunostaining and is a specific example of immunohistochemistry that makes use of fluorophores to visualize the location of the antibodies.
实验步骤
B or T cells in suspension, adherent cells on chambered coverglass or chamberslides, cryostat sections of unfixed, OCT embedded tissue:
1. Wash cells 1x cold RPMI (no wash for cryostat sections).
2. Fix 20 min 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4, 0.03M sucrose on ice. **
3. Wash 2x PBS/1%BSA. From now on everything can be at room temp, or on ice.
4. Permeabilize 5 min RT in 0.2% saponin, PBS, 0.03M sucrose, 1% BSA.
5. Wash 1x PBS/BSA.
6. Block 15 min 5% normal goat serum (NGS) in PBS/BSA.
7. Wash 1x PBS/BSA.
8. Diluted in PBS/BSA 60 min RT; 100 l per tube or section.
9. Wash 3x PBS/BSA.
10. Block 15 min 5% NGS in PBS/BSA.
11. Wash 1x PBS/BSA.
12. Diluted in PBS/BSA 30 min RT; 100 ul per tube or section.
13. Wash 3x PBS/BSA; (wash 1x in PBS/BSA then 2 x 10 min in Molecular Probes SlowFade Light buffer if using Slow Fade Light S-7461 to coversip); pellet cells and put up in two drops of Molecular Probes Slowfade Light antifade medium. Pipet about 15 ul on slide and coverslip. For chambered coverglass, just put two-three drops in each chamber after wash.
注意事项
1. When looking at B cells (they are relatively fragile) I have tried to support the coverslip with EM grids on two sides, 12 um diameter latex beads from Sigma (LB-120) mixed with the final cell suspension in antifade medium, and have tried #1 coverslips instead of #1.5. Using #1 coverslips has given me the fewest smashed cells. I now use an inverted scope and pipet the cells onto coverslip chamber slides (Lab-Tek #136439) in antifade medium so no worries about flattened, smashed cells.
2. To look at cytoskeleton I have fixed adherent cells on chambered coverslip slides and cryostat sections with ice cold methanol: acetone 1:1 for 10 minutes, then in ice cold ethanol: acetic acid 95:5 for 10 minutes, then wash in saline. Block and label as above steps 6-13.
3. Vectashield (Vector Laboratories H-1000 and H-1200 with DAPI) antifade medium gives better antifade protection than SlowFade Light but is hard on the cells; I see lots of membrane blebbing on both fixed B cells in suspension and on fixed adherent melanoma cell lines. Sometimes the problem is worse than others; sometimes with Slowfade Light I see blebbing. On tissue sections which can be allowed to air dry, Molecular Probes ProLong Antifade Kit (P-7481) is the best.
4. 1% Triton-X completely destroyed fixed B cells lines in my hands. 0.5% saponin as above does a good job of permeabilizing most of the cells and leaving them structurally intact.
5. Using 50mM NH4Cl in PBS or 0.15 M glycine in PBS to "quench" cells after paraformaldehyde fixation had absolutely no effect. I do not use either anymore.
6. Coating slides with poly-L-lysine (or using Sigma Poly-Prep slides P-0425) to make these cells in suspension stick to the slide did not help - they stuck maybe a bit but the coated slides picked up the secondary fluorescent tag somehow and were noisy. If you need a coated slide for your work use VWR Superfrost Plus #48311-703; sections stick throughout the labeling process and the slide does not give a noisy background.
7. Cells are fragile; handle gently even when fixed; do not vortex them. I routinely run cells up in Falcon #2054 tissue culture tubes, not microfuge tubes, and use low speed spins to pellet - just enough to get the cells down in 3-5 min. This way the cell "pellet" is always soft and easy to resuspend with gentle swirling. On chambered coverglass, I make sure that the addition and removal of solutions is done gently in the corner of the chamber so no cells are hit with a stream of any solution.
